Imaizumi K, Shih JY, Farris HE.

P20RR016816 NCRR NIH HHS , P30 GM103340 NIGMS NIH HHS , P20 RR016816 NCRR NIH HHS

	Figure 1. Development of spontaneous Ca2+ events in Xenopus tectum depends on stage but not visual-experience.(A) Movie frame of optic tectum stained by OGB-1AM. Spontaneous Ca2+ events were assessed in the neuropil (Np; surrounded by a dashed line). C: cell body layer, V: ventricular layer, R: rostral, L: lateral. (B) Example traces of spontaneous Ca2+ events in tecta reared under a control 12/12 hours light/dark cycle (CTRL: left column) and a dark condition (Dark: right column) at different developmental stages. (C–D) Analysis of event frequency and magnitude of spontaneous activity. Within-stage comparisons between the two rearing conditions were performed by unpaired t-test (left column). Between-stage comparisons after pooling the two rearing conditions were performed by Fisher's protected least significant difference test (right column). (C) Mean ± S.D. of event frequency in each groups is; 2.7 ± 1.0 (CTRL) vs. 3.9 ± 0.8 (Dark) at stage 45/46, 3.8 ± 1.0 vs. 3.7 ± 1.2 at stage 47/48, and 2.5 ± 1.0 vs. 2.7 ± 1.0 at stage 49/50. (D) Mean ± S.D. of event magnitude in each group is; 12.2 ± 8.1 (CTRL) vs. 15.1 ± 7.6 (Dark) at stage 45/46, 24.3 ± 9.1 vs. 28.9 ± 6.8 at stage 47/48, and 25.8 ± 10.8 vs. 16.2 ± 11.4 at stage 49/50. (E) Coefficient of variation (CV) of inter-event intervals (IEIs) of spontaneous activity. Mean ± S.D. of CV of IEI in each group is; 0.56 ± 0.14 (CTRL) vs. 0.53 ± 0.18 at stage 45/46, 0.48 ± 0.08 vs. 0.59 ± 0.18 at stage 47/48, and 0.59 ± 0.15 vs. 0.64 ± 0.12 at stage 49/50. Sample size at each stage in the two different rearing conditions is noted in (E). *: p<0.05, **: p<0.01, ***: p<0.001. Error bars are S.E.M.
	Figure 2. Experience-dependent synchrony in spontaneous Ca2+ events between neurons.(A–C) Examples from two tecta at stage 48 that varied in spontaneous Ca2+ event synchrony (left column: control light/dark; right column: dark-reared). (A) Raster plots of spontaneous Ca2+ events in single neurons and the neuropil (Np) illustrated by black and red dots, respectively. (B) Example traces of 13 single neurons and the neuropil shown in (A). (C) Matrix of maximum correlation coefficient (MCC) values for all cell pairs (including the neuropil) in cross correlation functions (CCFs). Insets: Examples of CCFs. Peak is MCC. A dashed line is 99% confidence threshold of CCFs. Time scale is between −100 s and 100 s. Medians of MCC values: 0.41 (left), 0.71 (right). (D) Comparisons of MCC values under different rearing conditions and at different stages. Box plots illustrate medians (thick horizontal lines), quartiles (box heights), 10th and 90th percentiles (error bars), and the outliers (circles). Statistical comparisons were performed by a resampling test. Pair numbers are illustrated below each distribution (bottom left). For multiple comparisons (right), p values were adjusted by Bonferroni correction. *: p<0.05, ***: p<0.001, ****: p<0.0001.
	Figure 3. Synchronized spontaneous activity between neurons.Ca2+ imaging was made by a faster frame speed (20 fps). (A) Ca2+ traces in seven neurons and the neuropil (Np). Gray lines are raw traces. Black and red lines are running average traces for illustration. (B) Maximum correlation coefficient (MCC) values for all pairs in cross correlation functions (CCFs) in a matrix. Cells are approximately aligned based on the distance along one direction. Inserts: Examples of CCFs for two pairs. Horizontal dashed lines illustrate the 99% of confidence threshold. A vertical dashed line is at a 0 s time lag.
	Figure 4. Neural transmission efficacy from the input (the neuropil) to the outpur (somata) layer is computed by mean maximum correlation coefficient (MCC) between the neuropil and cells.Within-stage comparisons between two rearing conditions were performed by unpaired t-tests (left column). Between-stage comparisons after pooling the two rearing conditions were performed by Fisher's protected least significant difference test (right column). Error bars are S.E.M. No significant difference suggests efficient neural transmission regardless of rearing conditions and developmental stages. Sample size at each stage in the two different rearing conditions is noted (left panel).
	Figure 5. Manipulation of input to the optic tectum.(A–D) Effect of enucleating the contralateral eye on spontaneous activity. Spontaneous Ca2+ events were assessed in a large area including the cell body layer and the neuropil. (A) Two examples of spontaneous Ca2+ events before (left) and after enucleation (right). (B–D) Event frequency, magnitude, and CV of IEIs of spontaneous activity before and after the enucleation. Data are normalized to the values before the manipulation. Comparisons were performed by paired t-tests in 10 tecta. (E–H) Effect of cutting the connection from the hindbrain to the tectum. (E) Two examples of spontaneous Ca2+ events before (left) and after cutting the connection between the tectum and the hindbrain (right). (F–H) Event frequency, magnitude, and CVs of IEIs of spontaneous activity before and after cutting the connection. *: p<0.05. (I–L) Effect of enucleating the contralateral eye and cutting the connection from the hindbrain to the tectum. (I) Two examples of spontaneous Ca2+ events before (left) and after the paired manipulations (right). (J–L) Event frequency, magnitude, and CVs of IEIs of spontaneous activity before and after the paired manipulations. ***: p<0.001. In (L), two cases that lost spontaneous activity after the manipulations were not included. Error bars are S.E.M. Colors in (A, E, and I) correspond to the same tecta in (B–D, F–H, and J–L). HB: hindbrain.
	Figure 6. Contrast between local non-neuronal and global neuronal synchronization.(A) ROIs of neurons (yellow circles), non-neuronal cells (blue circles) and neuropil (red dashed line) on average of three movie frames. Yellow dashed circles illustrate ROIs of neurons without spontaneous activity. Blue filled circles represent seven non-neuronal cells used in (B, D, and E). (B) Raster plot of spontaneous Ca2+ events of 62 neurons (gray), the neuropil (red), and 61 non-neuronal cells (black and blue). Note the global structure of spontaneous Ca2+ events in neurons and the neuropil and lack of that in non-neuronal cells. (C) MCC values of CCFs for all pairs of single neurons and the neuropil (top) and non-neuronal cells (bottom). Medians of MCC values: 0.48 for neurons and neuropil, 0.44 for non-neuronal cells. (D) Spontaneous Ca2+ traces of two neurons (gray), the neuropil (Np) (red), and 12 non-neuronal cells (black and blue). Spontaneous Ca2+ traces (blue) in seven non-neuronal cells were analyzed for local interactions. (E) Assessing local interactions among seven non-neuronal cells were performed by CCF analysis. Insets illustrate seven CCFs to show complex interactions.

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